Alphaviruses including Venezuelan (VEEV), eastern (EEEV) and western equine encephalitis viruses (WEEV) are highly developed agents of biological warfare and terrorism (BWT) and important, naturally emerging zoonotic viruses. Current biodefense against these viruses is inadequate, and widespread morbidity and mortality could be inflicted upon exposure of civilians or military personnel. Effective, licensed vaccines, critical first lines of defense and important tools for biodefense research, are badly needed. We will exploit recent advances in alphaviral genetics and vaccine design, as well as the unique alphavirology expertise within our regional group, to develop safe and effective vaccines suitable for licensure. We will generate a set of vaccine candidates against VEEV, EEEV and WEEV using 3 different strategies: 1) live-attenuated, chimeric Sindbis-based viruses expressing VEEV, EEEV and WEEV structural proteins; 2) replication-defective viral particles, based on the chimeric Sindbis-based genomes, that express heterologous envelope glycoproteins in vivo; and 3) chimeric Sindbis-based replicons that express in vivo the heterologous envelope glycoproteins, and also produce in vivo, virus-like particles lacking RNA. A major advantage of these chimeric systems, which have proved successful for flaviviruses, is that none will have the potential to generate or retain the encephalitic alphavirus parent virus or its complete genome. We will also combine these vaccines with several different adjuvants expressed from replicon systems to optimize the immune response. These candidate alphavirus vaccines will be evaluated in rodent models for safety, immunogenicity and protection against challenge including aerosol and mosquito infection. The live-attenuated and replicon-based alphaviruses developed in this project will take advantage of the greater antigen presentation, cell-mediated immunity and longer lasting antibody levels characteristic of exposure to replicating viruses. In addition to protection of civilian and military populations from an anticipated alphavirus BWT event, and protection of laboratory personnel doing critical BWT and public health research, the methods we will develop can be exploited to rapidly and efficiently develop new vaccines against newly recognized, emerging alphaviruses or an engineered alphavirus weapon. They will also be useful for protecting populations at risk of natural exposure to these zoonotic agents in many parts in the New World.